Electrophoretic destaining apparatus



Jan. 21, 1964 s. RAYMOND 3,118,829

ELECTROPHORETIC DESTAINING APPARATUS Filed March 28, 1963 2 Sheets-Sheet 1 FIG. 2

INVENTOR. SAMUEL RAYMOND 2; $40, Auk-5f;

L J ATTORNEY Jan. 21, 1964 S. RAYMOND ELECTROPHORETIC DESTAINING APPARATUS Filed March 28, 1963 FIG. 4

FIG. 3

2 Sheets-Sheet 2 IN VENTOR.

SAMUEL RAYMOND A TTOR NE Y5;

United States Patent 3,118,829 ELECTROPHDRETIC DESTAINING APPARATU Samuel Raymond, 341 S. 26th St., Philadelphia 3, Pa. Filed Mar. 28, 1963, Ser. No. 268,828 7 Claims. (Cl. 294-299) This invention relates to improvements in electrophoretic destaining apparatus of the type used with electrophoresis apparatus for the purpose of removing a certain amount of stain or dye from stained or dyed gel containing electrophoretically separated substances.

Electrophoresis, generally, relates to the separation of a complex substance into its component fractions by procedures based upon the migration or mobility of electrically charged fractions in a direct current electric field. With an electric field developed between two spaced electrodes at a substance placed therein, variously charged components or constituents of the substance move or migrate toward the respective electrodes of opposite charge and the respective components will move with different mobilities. This separation may take place on different support mediums such as a sheet of absorbent material like filter paper or on a permeable gel saturated with a buffer solution. Each component separated by the electrophoresis procedure is subject to a qualitative and quantitative analysis and therefore electrophoresis has provided a useful tool in laboratory analysis of various substances such as albumin, enzymes, hemoglobin, carbohydrates, blood serum, etc., as well as various inorganic ions. An example of prior art electrophoresis apparatus is my prior Patent 3,047,489, ranted July 31, 1962.

After the separation of the substances by electrophoretic migration, the slab containing the support medium and the separated components is stained and then a portion of the stain is removed to better provide a visible indication of the separated substances. In other words, it is conventional practice to follow electrophoretic migration by staining and then partial removal of the stain by washing or the like. This step is commonly called destaining and is provided to effectively develop the substances at the point to which they have migrated. Removal of the stain by washing or destaining is typically accomplished by washing with a solvent and usually takes a considerable amount of time, for example, from four to twenty-four or more hours.

My present invention is based partially on the discovery that instead of merely washing an electrophoretic sample slab, a DC. field may be applied across the slab in the washing solution to actively draw the dye or stain toward either the positive or negative electrode establishing the DC. field depending upon whether the dye molecules have a negative or positive charge. This transport of the dye molecules is much more rapid than simple diffusion into a washing solvent and permits reduction of the staining time.

in order to. provide optimum destaining, the electrodes are preferably positioned close to the entire surface of the sample gel slab. However, this creates problems of accidental contact between the slab and the sample and further creates problems of gas bubbles evolved from the electrodes which damage the gel slab if they contact it. It is one object of this invention to provide an electrode structure including two vertically positioned spaced foraminous electrodes such as mesh screens sand-wiching a gel slab therebe-tween. The vertical positioning of the electrodes permits the escape of gas generated by electrolysis in the solvent extracting solution while avoiding contact of such gaseous products with the surface of the gel. In addition, the gel slab is separated from each electrode by a porous material permeable to the liquid solvent and capable of passing electric current therethrough, but impermeable to gas bubbles at normal M 3,118,829 Ce Patented Jan. 21, 1954 atmospheric pressure. This porous material, such as sponge, prevents the gas bubbles liberated at the electrodes from reaching the surface of the gel where they would stick to the gel and result in a spotty decoloration or destaining. Additionally, the sponges prevent the gel from coming into direct contact with the electrode structure. Such direct contact between the electrode and the gel without the current first passing through the solvent results in localized overheating and burning and possibly localized chemical action on the electrophoretic gel sample slab. This contact, in any event, causes defects in the resulting electrophoretic pattern in the gel and is quite unacceptable.

It is a further object of this invention to provide a control for the electric field established across the sample gel slab while using only a simple, inexpensive and nonadjustable power supply. This is accomplished by con trolling the distance of separation between the electrodes either by setting the electrodes at a predetermined distance apart found by experiment to be the proper distance for use with a recommended destraining solvent; or providing adjustable means for supporting the electrodes various distances apart so that they may be adjusted in accordance with any chosen solvent which is used. Thus, the invention provides a means for controlling the power supply by the separation of the electrodes which in turn is related to the electrophoretic conductivity of the solvent used. By accomplishing the control based on the distance of electrode separation there is no necessity of having an expensive power controlling resistance or a variable transformer built in the power supply and therefore the apparatus is much more economical to operate as well as to make, since the electric power consumed within the electrode area is not wasted on the heating of a resistance in the supply and elimination of these extra components makes the apparatus more inexpensive to construct. Further, the power supply includes an isolating step-down transformer to separate the electrodes from ground and to prevent the user from getting a heavy shock from the apparatus.

An additional and most important object of this invention is to prevent remixing and recirculation of the liberated dye in the buffer solution within a destaining chamber. This object is accomplished by a unique construction of the destaining chamber for accomplishing streamlined or laminar flow of the buffer solvent stream. More particularly, the rate or flow is related to the passage of the stream through the destaining chamber so that streamlined flow picks up the liberated dye at the appropriate electrode and carries it directly to the outflow opening of the destaining chamber and discharges it directly onto a decolorizing adsorbent in a lower decolorizing chamber. By preventing remixing and recirculation of the dye bearing solvent within the destaining chamber, the efiiciency of the apparatus is greatly improved and the time required for destaining is reduced considerably. The unique construction of the destaining chamber with the electrode assembly therein provides that the inflow stream enters near the top of the destaining chamber and sweeps across the face to one electrode and underneath the electrode assembly then up across the face of the opposite electrode and out through an outlet opening chamber directly to the decolorizing chamher. After decolorizing, the solvent butter solution is recirculated.

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of a preferred embodiment of the invention, as illustrated in the accompanying drawings.

In the drawings:

FIG. 1 is a perspective view of the assembled apparatus 3 of this invention and a circuit diagram of the power supply of this invention;

FIG. 2 is a perspective view from another side of the apparatus with the electrode assembly removed;

FIG. 3 is a top plan view of the apparatus and the destaining chamber;

FIG. 4 is a sectional elevation view taken along line 44 of FIG. 3;

FIG. 5 is a perspective view of the electrode assembly when opened.

Referring now to the drawings, the electrophoretic destaining apparatus is indicated generally at 10. The apparatus is suitably supported as a self-contained unit from a base 12 which in turn carries a vertical support plate 14. Upstanding side walls 15, 16, 17 and 18 form together with the base an enclosed liquid holding tank 19. A horizontal floor 2t divides the tank 19 effectively into an upper destaining chamber 22 and a lower decolorizing chamber 24. The components forming the chambers may be constructed of machinable electrically insulating clear plastic such as Lucite. The device may also have a top cover, which is not shown in the drawings for the sake of clarity. Further, the upstanding side walls 15, 16, 17 and '18 may be horizontally divided immediately below floor 253 so as to provide two separate tanks, namely upper destaining tank or chamber 22 and lower decolorizing tank or chamber 24.

A'n electric motor 26 supported from vertical support plate 14 drives a pump 28. The pump is connected to a suction or inlet tube 30 which tube has its other end connected at 31 to the sidewall 18 of the lower dccolorizing tank 24. A pump outlet tube 32 is connected to the upper side wall 18 at the upper portion of the destaining tank 22 at connection 33.

The upper dcstaining chamber 22 is provided in one Corner with an angle-piece 34 forming a darn which has holes 36 therein for the flow of liquid solvent. There is also a hole 38 in the floor 20 of the upper destaining chamber 22; and the dam 34, holes 36 and hole 38 provide an outlet passage 39 for the solvent butter solution.

Below the floor 26 there is a V-shaped trough 40 having holes 42 therein for distributing circulated solvent solution over the top of a bed of adsorbent 44 which is preferably decolorizing charcoal for the purpose of removing the dye from the solvent and allowing it to be recirculated.

Within the lower decolorizing chamber 24 there is a bracket 46 for containing a filter sponge 48, to prevent the charcoal from being circulated with the solvent. At

the bottom corner of the filter sponge there is a return channel 50 for the return and recirculation of solvent through pump inlet tube 30.

An electrode assembly 52 may be removably positioned across the upper destaining tank 22. The electrode assembly includes a positive electrode 54 and a negative electrode 56 and the position of these electrodes relative to solvent inlet 33 and outlet passage 39 for the destaining tank can be switched by removing the electrode assembly 52 and turning it around. Since each electrode assembly is identical, only one will be described. The electrode assembly includes a contact tab or connection 58 which is electrically connected to a grid or wire mesh 69 extendling over a large surface so as to apply a uniform field to :a gel sample slab 72. The screen 69 and contact 58 are contained within an insulating holder 62 which is hinged by hinges 64 to a similar holder for the other electrode. As shown in FIG. 4, the holder may have spacers 65 on the bottom surface thereof to space the bottom of the holder above the top of floor 2t) and thereby allow the solvent to pass under the electrode assembly 52 as 'well as through separating sponges 63 which are held within a lip of flange 66 of the holder 62. The separating sponges 68 are adapted to separate the electrode 60 from a slab 72 of prestained gel containing the electrophoretically separated components of a substance. The

sponges allow the current to be applied through the solution to the gel while preventing the electrodes coming in contact with the gel and while preventing bubbles liberated at the electrode from contacting the gel. The arrangement also allows for streamline flow of the solvent past the surface of the electrodes and the flow is streamlined or laminar as it passes outwardly.

The power supply for supplying the electrodes with direct current is shown in FIG. 1. It may conveniently include an electrical cord 71 for plugging into any conventional 110 volt A.C. house circuit. The power supply is shown in dotted line 7 0 representing a housing which may, if desired, be supported from vertical support plate 14 on the side thereof opposite motor 26. Suitable control switches 73 and 75 may be provided for controlling the circuit to the electrodes and to the pump motor as shown in FIG. 1. The circuit to the electrodes includes an isolating step-down transformer 74 and a full wave bridge rectifier 76. The transformer 74 isolates the electrodes from ground to eliminate the possibility of receiving substantial shocks from the use of the device. An indicator lamp 77 may be provided to indicate when the DC. field is applied to the electrodes.

It is believed the operation of the apparatus is apparent from the foregoing, but a brief synopsis of the operation will now be given.

After electrophoretic separation is performed on a substance by migration of the components on a support me dium such as a gel or the like, the slab 72 containing the results of such electrophoretic migration is stained or dyed with a suitable stain or dye as is known in the art, for example amidoblack. The stain completely darkens the slab so that the results of the electrophoretic migration cannot be seen. It is then necessary to remove a selected portion of the dye so that the separated components can be seen. This is accomplished by placing the slab 72 between the separating sponges 68 and also between the spaced electrodes 54 and 56 which are constructed as mesh screens 60. The electrode assembly 52 is then placed in the upper destaining tank 22. The lower destaining tank 44 has been previously filled with adsorbent charcoal and the tanks contain a quantity of solvent for recirculation past the electrodes for removing the dye. The electrode assembly is placed in the destaining tank in such a manner that the electrode at which the dye is liberated is closest to the outlet passage 39. For example, with amidoblack the dye will be attracted to the positive electrode and will be swept by the solvent and buffer solution over darn 1 and through outlet 39 for decolorizing and recirculation.

With the switches 73 and 7S closed, a DO field will be applied to the electrodes 54 and 56 and the motor 26 will drive the pump 28 circulating the bulfer and solvent. The passage of the solvent will be from the pump 28 through outlet 32 into the top of destaining tank 22 from whence it will flow downward over the surface of the negative electrode. The solvent will flow in streamline flow under the electrode assembly and up the opposite face thereof and will pick up the dye liberated on electrode 54, for example. Then the flow of the solvent will be through holes 36 down passage 38 and into trough 40. The solvent containing the liberated dye or stain then passes out holes 42 of trough 40 and drops over the bed of charcoal 44 in the decolorizing tank 24. The solvent then flows through the charcoal and the dye or stain is removed from the solvent by adsorption on the surface of charcoal as is known in the art. The solvent then flows through the filter sponge 48 which keeps the charcoal 44 out of the recirculating stream and the decolorized solvent or buffer is recirculated through pump inlet tube 38 and again through the circuit as described.

While the invention has been particularly shown and described with reference to a preferred embodiment thereof, it will be understood by those skilled in the art that various changes in form and details may be made 5 therein without departing from the spirit and scope of the invention.

What is claimed is:

1. An electrophoretic destaining apparatus comprising in combination;

(a) an upper destaining tank,

(1)) a lower decolorizing tank juxtapositioned below the upper destaining tank,

(c) an outlet passage providing fluid communication between one portion of the upper destaining tank and the lower decolorizing tank,

((1) another fluid passage providing fluid communication from the lower decolorizing tank back up to the upper destaining tank at an inlet passage on a side thereof opposite the outlet passage,

(e) a motor drive pump for circulating a solvent liquid from the lower to the upper destaining tank through the outlet passage, to the lower decolorizing tank, and back through the recirculating passage,

(1) a decol'orizing adsorbent in the lower tank for adsorbing the stain from the circulated solvent,

(g) an electrode assembly including positive and negative electrodes, the assembly being vertically positioned across the upper tank between the inlet and outlet passages,

(It) means affording a restricted fluid flow passage past the so positioned electrode assembly to afford streamline flow of the recirculated liquid,

(i a pair of foraminous electrodes vertically supported in spaced relation in the electrode assembly,

(j) a pair of porous liquid permeable but substantially gas impermeable blocks positioned to sandwich an electrophoretic slab to be destained and in turn be sandwiched by the pair of vertical foraminous electrodes, and

(It) means for supplying a predetermined potential direct current to the electrodes and across the so positioned electrophoretic slab.

2. A11 electrophoretic destaining apparatus as defined in claim 1 wherein the two tanks have common upstanding side walls with a generally horizontally extending floor dividing the two juxtapositioned tanks.

3. An electrophoretic apparatus as defined in claim 2 wherein the outlet passage is in one corner of the upper 6 destaining tank and it comprises an angle-dam which, together with the corner of the tank, surrounds a hole in the floor, and the inlet for the recirculated liquid is in an opposite corner.

4. An electrophoretic destaining apparatus as defined in claim 3 further comprising a leak trough extending up to the floor from the outlet hole to distribute recirculated liquid over the decolorizing adsorbent means in the lower decolorizing tank.

5. An electrophoretic destaining apparatus as defined in claim 4 wherein decolorizing means includes decolorizing charcoal, and further comprising a filter in the lower decolorizing tank to keep the charcoal in the tank and out of the recirculated liquid solvent.

6. An electrophoretic apparatus as defined in claim 1 wherein the electrode assembly is a hinged structure with one leaf of the hinge structure carrying one electrode, and the means affording restricted passage comprising spacers for spacing the electrode assembly of the bottom or" the tank, and wherein the electrode assembly is positioned diagonally across the tank from corner to corner so as to be in the path of liquid flowing from the inlet passage to the outlet passage of the upper destaining tank.

7. An electrophoretic destaining apparatus as defined in claim 1 wherein the means for supplying a predetermined potential DC. to the electrode applies a constant DC. potential and comprises an isolating stepdown trans former and a rectifier, and wherein the current across the slab between the electrodes is dependent upon the spacing of the electrodes.

References Cited in the file of this patent UNITED STATES PATENTS OTHER REFERENCES Ferris et al.: American Journal of Chemical Pathology, Electrophoresis of Serum Proteins in Acrylamide Gel, vol. 38, No. 4, pages 383-387, October 1962. 

1. AN ELECTROPHORETIC DESTAINING APPARATUS COMPRISING IN COMBINATION; (A) AN UPPER DESTAINING TANK, (B) A LOWER DECOLORIZING TANK JUXTAPOSITIONED BELOW THE UPPER DESTAINING TANK, (C) AN OUTLET PASSAGE PROVIDING FLUID COMMUNICATION BETWEEN ON EPORTION OF THE UPPER DESTAINING TANK AND THE LOWER DECOLORIZING TANK, (D) ANOTHER FLUID PASSAGE PROVIDING FLUID COMMUNICATION FROM THE LOWER DECOLORIZING TANK BACK UP TO THE UPPER DESTAIONING TANK AT AN INLET PASSAGE ON A SIDE THEREOF OPPOSITE THE OUTLET PASSAGE, (E) A MOTOR DRIVE PUMP FOR CIRCULATING A SOLVENT LIQUID FROM THE LOWER TO THE UPPER DESTAINING TANK THROUGH THE OUTLET PASSAGE, TO THE LOWER DECOLORIZING TANK, AND BACK THROUGH THE RECIRCULATING PASSAGE, (F) A DECOLORIZING ADSORBENT IN THE LOWER TANK FOR ADSORBING THE STAIN FROM THE CIRCULATED SOLVENT, (G) AN ELECTRODE ASSEMBLY INCLUDING POSITIVE AND NEGATIVE ELECTRODES, THE ASSEMBLY BEING VERTICALLY POSITIONED ACROSS THE UPPER TANK BETWEEN THE INLET AND OUTLET PASSAGES, (H) MEANS AFFORDING A RESTRICTED FLUID FLOW PASSAGE PAST THE SO POSITIONED ELECTRODE ASSEMBLY TO AFFORD STREAMLINE FLOW OF THE RECIRCULATED LIQUID, (I) A PAIR OF FORAMINOUS ELECTRODES VERTICALLY SUPPORTED IN SPACED RELATION IN THE ELECTRODE ASSEMBLY, (J) A PAIR OF POROUS LIQUID PERMEABLE BUT SUBSTANTIALLY GAS IMPERMEABLE BLOCKS POSITIONED TO SANDWICH AN ELECTROPHORETIC SLAB TO BE DESTAINED AND IN TURN BE SANDWICHED BY THE PAIR OF VERTICAL FORAMINOUS ELECTRODES, AND (K) MEANS FOR SUPPLYING A PERDETERMINED POTENTIAL DIRECT CURRENT TO THE ELECTRODES AND ACROSS THE SO POSITIONED ELECTROPHORETIC SLAB. 